Analysis of the ¹H‐NMR Chemical Shifts of Cu(I)‐, Cu(II)‐ and Cd‐Substituted Pea Plastocyanin

Abstract

To compare cadmium-substituted plastocyanin with copper plastocyanin, the 'H-NMR spectra of CuI-, Curl-and Cd-plastocyanin from pea have been analyzed. Full assignments of the spectra of CuIand Cd-plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd.For CuII-plastocyanin the resonances of 72% of the protons observed in the Cul form have been identified. Protons within =0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9 -1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both Cur-and Cd-plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII-plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI-plastocyanin, indicating that the CuII-plastocyanin structure, in as far as it can be observed, resembles Cd-rather than CuI-plastocyanin. In a single stretch of residues (64-69) chemical shift differences remained between all three forms after correction.The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0-0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9-1.7 nm), in which protons can be observed and show pseudocontact shifts.Keywords: plastocyanin ; chemical shift; paramagnetic ; azurin ; cadmium.It is concluded that Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plastocyanin.Plastocyanin is a small (10.4 kDa) type-I copper protein that functions as a soluble electron carrier in the lumen of thylakoid vesicles in chloroplasts. It shuttles electrons from the cytochrome lf complex to photosystem I (PSI), both of which are located in the thylakoid membrane. There is much interest in the question of how plastocyanin interacts with its redox partners. The protein has two obvious potential sites for electron transfer. First, the so-called hydrophobic patch at the top or north side of the protein in the conventional representation (Fig. 1) which provides the shortest route for the electron to reach the copper atom, via the ligand H87. Secondly, the acidic patch (at the east side) which requires a longer pathway for electron transfer (via ligand C84 and surface residue Y83) but contains two clusters of negative charges, which are potentially favourable for electrostatic interaction between plastocyanin and the redox partners.A variety of tec...

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

“…To elucidate the PSI binding mode of Pc in more detail, the Pc-PSI interaction was further investigated under conditions where the interactions on the hydrophobic patch of Pc were perturbed by cadmium substitution. A comparison of the 1 H-15 N shift correlation spectra of uniformly [ 15 N]-labeled Pc and cadmium-substituted Pc (Cd-Pc) revealed that the signals that exhibited significantly different chemical shifts were all those from the hydrophobic patch residues, consistent with the previous report for Pcs from different species (Ubbink et al, 1996), suggesting that the Cd-Pc is correctly folded (see Supplemental Figures 6A and 6B online). Although the distance between the amide proton of Gly-8 and the Hb of Phe-14, which are located at the stem of the loop1, is 9.5 Å in the structure of reduced Pc, a nuclear Overhauser effect, which is usually observed for hydrogen atom pairs with <5 Å distance, was observed between the corresponding hydrogen atoms in CdPc, suggesting that the conformation of loop1 in the hydrophobic patch is perturbed by the cadmium substitution.…”

“…The substitution of the copper ion of Pc with a cadmium ion reportedly shifts the hydrogen bond network in the hydrophobic patch to a configuration resembling that in the oxidized Pc, without affecting the conformations of the other regions, including the acidic patch (Ubbink et al, 1996). To elucidate the PSI binding mode of Pc in more detail, the Pc-PSI interaction was further investigated under conditions where the interactions on the hydrophobic patch of Pc were perturbed by cadmium substitution.…”

“…The Cd-Pc was prepared according to the previously published procedure (Ubbink et al, 1996) and was purified by Hiload 26/60 Superdex 75 prep grade chromatography and Resource Q chromatography.…”

Backbone Dynamics of Plastocyanin in Both Oxidation States

“…7 shows that 1 H and 15 N chemical shift differences are found in the regions encompassing all four northern loops, whereas none of the regions around the southern loops are involved. Whereas 15 N chemical shift differences have been discussed in terms of very minor steric/ electronic variations (69 -75), the 1 H chemical shift differences can be tentatively ascribed to modest structural modifications consequent to a redox state change of the copper ion, as also found in a similar analysis for pea plastocyanin (72).…”

On the use of pseudocontact shifts in the structure determination of metalloproteins

“…In particular, the amide protons located in the four loop regions surrounding the metal site undergo significant changes in chemical shift, as was also observed previously in the case of pea plastocyanin. 31 These changes clearly demonstrate the importance of choosing a proper diamagnetic reference. …”